For most therapeutic drugs administered by intra-arterial or intravenous routes the intended site of molecular activity lies outside the vasculature. For drugs administered via the airways, the intended site of activity normally is beyond the first cellular barrier of alveolar, bronchiolar or tracheal epithelia. In both cases, there is an endothelial or epithelial barrier which must be crossed before the drug can mediate its effect.
For small lipophilic drugs, there appears to be a paracellular route between the tight junctions of the barrier cells. However, for hydrophilic drugs and larger macromolecular active agents, such as peptides, proteins, genes or anti-sense nucleotides, the only route across the barrier is through the cells. This poses a particular problem for drugs administered intravenously which have exceedingly short half-lives due to rapid degradation or first pass clearance by the liver. In order to maintain therapeutic levels in balance with such excretion and degradation, large doses or infusions are often necessary. Thus, there is clearly a need in the art for more rapid mechanisms for delivering drugs across cellular barriers.
There have been numerous reports of specific receptors which mediate endocytotic events, where a ligand binds to the receptor and is then internalized, complexed to the receptor, by a process similar to pinocytosis. This involves invagination of the cell membrane in the region of the ligand receptor complex and then release of the ligand into the cell by a process which is not fully understood. Numerous endocytotic receptor systems have been reported including LDL, insulin, epidermal growth factor, insulin-like growth factor and tPA-PAI-I (hybrid molecule).
Transcytosis entails invagination and vesicle formation around a ligand receptor complex, followed by transcytotic passage with release by a reverse invagination process at the basolateral membrane. Monoclonal antibodies to the transferrin receptor have been conjugated with toxins, so that they can undergo transcytosis, across blood-brain endothelia. However, there is a continuing need in the art for agents capable of delivering or enhancing passage of drugs by receptor-mediated transcytosis across cellular barriers other than blood-brain endothelia, such as endothelia of the vasculature, alveolar epithelia, and peritoneal mesothelia.
The GP60 receptor, also referred to as albondin, is one of several albumin-binding proteins reported in the literature (Schnitzer and Oh, J. Biol. Chem. 269(8):6072-6082 (1994)). Others include SPARC (serum protein, acidic, rich in cysteine), oesteonectin or basement membrane protein 40, GP30, GP18 and GP60. SPARC and oesteonectin are extra-cellular proteins. GP60 shares some homology with SPARC as determined using anti-SPARC antibodies (Schnitzer and Oh, Am. J. Physiol. 263:H1872-H1879 (1992)).
GP18 and GP30 are membrane glycoproteins found in a variety of cell types but are particularly prevalent in the macrophage (Schnitzer et al, J. Biol. Chem. 267: 24544-24553 (1992)). GP18 and GP30 are the so-called "scavenger receptors" responsible for mediating removal of oxidized, glycated or adduced forms of albumin by endocytosis and are thus believed to play a role in albumin catabolism for a wide variety of organs (Schnitzer and Bravo, J. Biol. Chem. 268(10):7562-7570 (1993)).
In contrast to GP18 and GP30, the GP60 receptor has found to be expressed exclusively in continuous endothelia of the vasculature (Schnitzer, Am. J. Physiol. 262:H246-H254 (1992)), in alveolar epithelia (Kim et al, Am. J. Resp. and Crit. Care Med. 151:A190, (1994) and inferentially in peritoneal mesothelia (Gotloib and Shostak, Kidney International. 47:1274-1284 (1995)). GP60 is particularly abundant in the microvessel endothelia and is, interestingly, absent from the blood-brain barrier, where little albumin flux is observed (Rousseaux et al, Methods in Enzymology 121:163 (1986)). It has been shown that polyclonal antibodies to endothelial GP60 also bind alveolar epithelial GP60 (Kim et al, supra). The GP60 receptor has been implicated in receptor-mediated transcytosis of albumin across epithelia and endothelial cell barriers (Kim et al, supra; Tirrupathi et al, Molecular Biology of the Cell 4 (Supp):338a, Abstract No. 1964 (1993)).
The GP60 amino acid sequence is known in the art (Yamauchi et al, Biochem. Biophys. Res. Comm. 146:1485 (1987)).